The field of bioengineering combines the methods of engineering with the principles of life science to understand the structural and functional relationships in normal and pathological organs. A goal of bioengineering is also the development and ultimate application of biological substitutes to restore, maintain, and improve organ functions, and thus, improve quality of life.
The eye is the organ of vision. A space called the anterior chamber is located in the front part of the eye. It is filled with a clear, watery fluid called aqueous humor. This fluid is continuously produced inside the eye by the ciliary body. It is different from tears, which are produced by glands outside of the eye and moisten the outer surface of the eyeball. This intraocular fluid flows out through the pupil, into the anterior chamber and exits the chamber at the angle where the iris and cornea meet. The majority of this fluid in humans flows through a structure called the trabecular meshwork (TM) into a channel called Schlemm's canal (SC). It then flows into 8-13 collector channels, which communicate with the venous system, thus eventually being absorbed into the bloodstream.
The human trabecular meshwork (HTM) is a spongy meshwork of drainage canals composed of collagenous and elastin extracellular matrix (ECM) on which reside cells specialized in the production and maintenance of this matrix, phagocytosis of debris, and regulation of aqueous humor flow. Cells in the wall of SC that is adjacent to the HTM also provide resistance to this flow. Proper drainage keeps the eye pressure at a normal level. The production, flow, and drainage of this intraocular fluid is an active continuous process that is needed for the health of the eye.
The inner pressure of the eye (intraocular pressure, IOP) depends on the rate of elimination of aqueous humor mostly through the HTM as the rate of production is relatively constant. If the eye's drainage system is working optimally, then any rise in IOP is prevented since the aqueous humor can drain out into the bloodstream. While IOP can vary at different times of the day, it is maintained within a narrow optimal range for normal individuals.
When resistance to outflow of aqueous humor rises because of disease, trauma to the HTM, or certain pharmacologic treatments, IOP increases above the normal range. High pressure may damage the sensitive optic nerve and result in vision loss. This condition is called glaucoma. In the majority of cases of open angle glaucoma, the eye's TM drainage system becomes “clogged” so the aqueous humor cannot drain efficiently. As aqueous outflow is impeded, IOP rises within the eye.
The eye has approximately one million nerve fibers that originate in the retina and form the optic nerve. These nerve fibers meet at the optic disc. As IOP builds up within the eye, it damages the nerve fibers and they begin to die. As the nerve fibers die, the optic disc begins to hollow and develops a cupped or curved shape. If the IOP remains too high for an individual, for too long, then it could lead to optic nerve damage and result in vision loss.
The most recent statistics by the World Health Organization have placed glaucoma as a leading cause of irreversible blindness worldwide, affecting nearly 70 million people. In addition, with the world's population aging, it is expected that the number of people affected by glaucoma will increase dramatically. Currently, the only treatment for glaucoma is lowering IOP, which is the only modifiable risk factor for glaucoma.
The HTM is an intricate 3-D structure, consisting of HTM cells and their associated ECM, including interwoven collagen beams and perforated sheets composed of elastin arranged in a laminar pattern. Glaucoma is thus linked to HTM structure and function where structural changes of the HTM likely affect tissue rigidity and biomechanical properties that influence its resistance to flow.
Current treatments of glaucoma involve lowering the IOP by means of decreasing aqueous humor production or increasing non-trabecular aqueous humor outflow. Few therapeutic agents primarily target the HTM. Of those few, in current clinical practice, only miotics (i.e. direct and indirect cholinergic agonists, e.g., pilocarpine) increase HTM outflow by contracting the ciliary muscle, the tendons of which “stretch” the TM to increase flow. The compound: latruncunlin-B (Lat-B), has recently gained interest as a potential novel glaucoma treatment. Lat-B increases aqueous humor outflow and decreases IOP by directly acting on HTM cells. Further advances in pharmacological treatment of glaucoma are currently limited by the lack of proper, efficient, in vitro models for the screening of new potential therapeutics.
While conventional HTM cell cultures may be useful for studying the biology of HTM cells, they are not suitable for evaluating the effects of medications on outflow facility (see, e.g., Koga et al., Exp Eye Res 82:362-70, 2006. Current outflow facility studies mainly rely on anterior segments of animal or human eyes (see, e.g., Ethier et al., Invest Ophthalmol Vis Sci 47:1991-8, 2006; however, the preparation of these perfusion systems is cumbersome, expensive, and not suitable for high-throughput screening. Therefore, there is a great need for a bioengineered, functional, in vitro HTM model for glaucoma drug screening.
Commercially available, porous membranes were tried for HTM cell culture, but with limited success (see, e.g., Perkin et al., Invest Ophthalmol Vis Sci 29:1836-1846, 1988). This is because these membranes either possess irregular pore structure or low porosity (e.g., 4-20%), which limits their performance for HTM cell growth and usefulness in perfusion experiments. In addition these membranes have not been adapted, suggested or used for high-throughput screening of medications. There is a need to overcome these disadvantages with an in vitro system that offers a new avenue for understanding the HTM physiology at the molecular and cellular level and testing pharmacological agents that affect trabecular outflow facility in humans. Recently, such a system was proposed to culture SC cells in a microfluidics-based hydrogel culture system that can be used to study the formation of giant vacuoles in SC cells (see, e.g., Vickerman et al., Invest Ophthalmol Vis Sci 51:E-Abstract, 5834, 2010). However, this system does not contain HTM cells, has not been used for study of pharmacologic agents, and would be difficult and very expensive to integrate in a high throughput system. In particular, a hydrogel-based system is not ideal for regulating flow and studying flow physiology at a stable condition.
Surgical glaucoma treatment attempts to either “stimulate” the TM to work better (laser trabeculoplasty) or altogether bypasses the TM, shunting the aqueous humor mostly to an extraocular space. Incisional glaucoma surgery currently consists of either trabeculectomy or seton surgery.
Trabeculectomy creates a fistula from the anterior chamber to the anterior subtenon's space. There aqueous humor is sequestered in cystic spaces and either gets absorbed in lymphatic vessels, enters episcleral veins or transudates through the conjunctiva. Trabeculectomy success depends in large part on appropriate management of the fibrotic (healing) response of each individual patient. Since this response is variable, surgical outcomes are often unpredictable. Even in cases where trabeculectomy is initially deemed successful, aqueous flow into the subtenon's space leads to progressive remodeling of the periocular and episcleral tissues. This remodeling ultimately leads to failure of surgery by limiting the area of diffusion of the aqueous thus increasing resistance to flow.
Seton surgery utilizes a prosthetic conduit (a silicone tube) to shunt aqueous humor to the posterior (>8 mm from the limbus) subtenon's space. The aqueous diffuses over the area of a variably sized plate that is attached to the tube. A fibrous capsule is formed by the host over the plate. The permeability of this capsule determines the rate of aqueous diffusion. Tube surgery is equally effective to trabeculectomy (see, e.g., Gedde et al., Curr Opin Ophthalmol 23:118-26, 2012). Its long-term failure is again the result of tissue remodeling of the plate capsule to make it progressively thicker and less permeable.
It is thus obvious that incisional glaucoma surgery is in a strict sense “non-physiologic”. Ideally, such surgery would bypass the defective/diseased TM/SC complex but would utilize the downstream physiological outflow path (SC, aqueous veins). Attempts to create such bypass with zero resistance to flow have in the long-term generally been unsuccessful to date. In the short term, they provide only small IOP decreases (see, e.g., Morales-Fernandez et al., Eur J Ophthalmol 22:670-3, 2012). In addition, since such attempts rely on complete bypass of the TM/SC complex, they can cause early hypotony, which can lead to significant vision-threatening complications. In addition, if the surgery is effective, the regulatory function of the TM/SC complex is lost.
Prior attempts to create a device that would utilize acellular micropatterned structures (see, e.g., Helies et al., J Fr Ophtalmol 21:351-60, 1998; Pan et al., Proc 28th IEEE EMBS Ann Int Conf, 2006) to act as HTM have failed to result in clinically useful devices because they rely on passive regulation of flow afforded by very narrow channels that over time get occluded by cells and debris in the aqueous humor in vivo, as well as by the fact that as with conventional glaucoma surgery, they shunt aqueous humor to the subconjunctival space, thus inducing a fibrotic response.
Thus the development of a bioengineered HTM that can replace the defective HTM in glaucoma would provide a novel and highly desirable way of understanding and treating the disease.